Kotaro Matsumoto

Detection of Anaplasma bovis and Anaplasma phagocytophilum DNA from Haemaphysalis megaspinosa in Hokkaido, Japan.

DNA from 101 ticks, including Haemaphysalis megaspinosa, Haemaphysalis douglasi, Haemaphysalis flava, Ixodes ovatus and Ixodes persulcatus, collected by flagging in a pasture in Hidaka district (Hokkaido, Japan) was examined for infection with Anaplasma bovis and A. phagocytophilum by species-specific nested PCR. Of the 48 H. megaspinosa nymphs, examined, 1 (2.1%) and 6 (12.5%) were positive for A. bovis and A. phagocytophilum, respectively. One of the 6 positive nymphs was dual positive for both pathogens. Of the 4 larval pools of H. megaspinosa examined, 1 pool was positive for both A. bovis and A. phagocytophilum. None of the samples of the other tick species and adults of H. megaspinosa showed any positive results. These results suggest that H. megaspinosa is a dominant vector tick species of both A. bovis and A. phagocytophilum for cattle in the pasture of Hidaka district, Japan.

Genetic diversity of Theileria orientalis in tick vectors detected in Hokkaido and Okinawa, Japan

In the present study, we investigated the possible tick vectors that can transmit Theileria orientalis in eastern Hokkaido, Japan. Questing ticks collected from three different districts, Taiki, Otofuke, and Shin-Hidaka, of Hokkaido included Ixodes persulcatus, Haemaphysalis megaspinosa, Haemaphysalis douglasi, and Ixodes ovatus, while all the ticks collected from Yonaguni island of Okinawa were identified as Haemaphysalis longicornis. When the ticks were screened by polymerase chain reaction (PCR) for T. orientalis, the parasite was commonly detected among all tick species. Genotype-specific PCR assays revealed that all tick species in Hokkaido were predominantly detected with type 2, while ticks collected from Okinawa (H. longicornis) were predominantly detected with type 1. Consistent with the genetic diversity of T. orientalis in ticks, genotyping PCR assays from cattle grazed in the same Hokkaido sampling locations identified type 2 as the most prevalent genotype. This study provides the first identification of I. persulcatus, H. megaspinosa, H. douglasi, and I. ovatus as possible tick vectors of T. orientalis, and finds that the variety of vectors apparently capable of transmitting T. orientalis is wider in Japan than expected. The authors suggest that tick control strategies should be modified in Hokkaido based on the seasonal activities of ticks identified in the present study.

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.

Molecular survey of bovine vector-borne pathogens in Cebu, Philippines

Vector-borne diseases (VBDs) continue to threaten the worldwide livestock industry, but comprehensive epidemiological surveys on such diseases have not been conducted in the Philippines. In the present study, we screened 408 bovine blood samples from 9 areas in Cebu, Philippines, for various VBD pathogens using specific PCR assays. The results revealed prevalences of 54.7, 15.4, 10.0, and 12.0% for Anaplasma spp., Babesia bigemina, Babesia bovis, and Trypanosoma (Tr.) theileri, respectively. In contrast, none of the samples were positive for Trypanosoma (Tr.) evansi, Theileria (Th.) orientalis, and Theileria (Th.) annulata. Mixed infections were observed in 24.2% of the samples tested. Phylogenetic analysis based on the 16S rRNA gene revealed that the Anaplasma spp. sequences from the present study were genetically close either to Anaplasma marginale or Anaplasma phagocytophilum. In addition, B. bovis RAP-1 and Babesia bigemina AMA-1 gene sequences were identical and monophyletic to other known B. bovis and B. bigemina sequences. On the other hand, Tr. theileri cathepsin-L like protein gene sequences shared 97.1-100% identities with those from the USA and Brazil and clustered within a single genotype in the phylogenetic tree. The molecular identification of several VBD pathogens in Cebu cattle calls for the implementation of control measures to prevent the spread of these pathogens to nearby localities or islands, and ultimately, economic losses to the Philippine economy.

PCR detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis.

ABSTRACT We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.

The first survey of Theileria orientalis infection in Mongolian cattle

In the present study, we have surveyed the presence of a bovine Theileria protozoan, Theileria orientalis, in Mongolian cattle and engorging tick populations from selected provinces and districts in Mongolia. The percentages of infection in the cattle and ticks ranged from 8.8 to 66.6 and from 3.7 to 73.3, respectively, on a per district basis. The genetic diversity of T. orientalis isolates was also studied, based on the protozoan gene encoding a major piroplasm surface protein (MPSP). At least five genotypes (types 1, 3, 5, 7, and N-3) of T. orientalis were found to be circulating among the Mongolian cattle and tick populations. In particular, types 3 and N-3 were common in most of the districts examined, while a strong geographical relationship among the genotypes was not detected in the present study. This is the first epidemiological report describing the presence of T. orientalis infection in Mongolian cattle.

Molecular Survey of Anaplasma and Ehrlichia Infections of Feral Raccoons (Procyon lotor) in Hokkaido, Japan

Infection by Anaplasma and Ehrlichia in feral raccoons (Procyon lotor) in Hokkaido, Japan, was examined by molecular methods. A polymerase chain reaction (PCR) screen for Anaplasmataceae, based on 16S rRNA, showed that 38 (5.4%) of 699 raccoons examined were positive. These 38 positive samples were examined for Anaplasma phagocytophilum, Anaplasma bovis, Ehrlichia chaffeensis, and Ehrlichia canis infection by species-specific nested PCR. Nested PCR results indicated that 36 of the 38 samples were positive for A. bovis. All 38 samples were PCR negative for A. phagocytophilum, E. chaffeensis, and E. canis. This is the first report of the detection of A. bovis in the peripheral blood of raccoons. A total of 124 raccoons were infested with ticks, including Ixodes ovatus, Ixodes persulcatus, and Haemaphysalis spp. The rate of A. bovis infection in raccoons infested with Haemaphysalis spp. (46.7%, 7/15) was significantly higher than that in raccoons without Haemaphysalis spp. infestation (3.7%, 4/109, p < 0.001). No significant differences were observed in A. bovis infection rates between raccoons infested with I. ovatus or I. persulcatus and those not so infested. A total of four ticks (two males and two nymphs) and one larval pools from four raccoons showed positive for A. bovis-specific nested PCR. This results support the correlation between the A. bovis infection of raccoons and Haemaphysalis infestation. In conclusion, raccoons could be possible reservoir animals for A. bovis, and A. bovis infection in raccoons may be related to infestation with Haemaphysalis spp.

Serum Thymidine Kinase Activity as a Useful Marker for Bovine Leukosis

Serum thymidine kinase (TK) activity has recently been evaluated as a serum marker for human and canine hematopoietic neoplasms. The purpose of the current study was to establish the significance of serum TK activity in the diagnosis of bovine leukosis. The discrimination value for TK activity was set at 5.4 U/l based on the receiver operating characteristic curve. In the group of clinically healthy cows, only 2 out of 83 cows (2.4%) had serum TK activity above the discrimination value. In contrast, 19 out of 20 cows (95.0%) with bovine leukosis showed serum TK activity above the discrimination value, although only 7 of 79 (8.9%) cows diagnosed with diseases other than bovine leukosis showed elevated serum TK activity. Thymidine kinase activities of all Bovine leukemia virus-positive cows with or without lymphocytosis were below the discrimination value. Sensitivity and specificity of measuring serum TK activity as a diagnostic tool for bovine leukosis was 95.0% and 95.9%, respectively. Results indicate that serum TK activity may be a marker for bovine leukosis.

Specific Molecular Detection of Anaplasma sp. Closely Related to Anaplasma phagocytophilum in Ixodid Ticks and Cattle in a Pastureland in Hokkaido, Japan

Recent molecular analyses of the Anaplasma sp. closely related to Anaplasma phagocytophilum (previously believed to be A. phagocytophilum) in Japan have clarified its distinct phylogenetic position. PCR methods relying on 16S rRNA- and P44/MSP2-based primers designed to detect this species have low sensitivity and specificity. In this study, a highly sensitive and specific nested PCR method using newly designed primers based on heat-shock operon gene (groEL) was developed to detect this species. The method was later used in an epidemiological study testing DNA samples from 85 Ixodid ticks (collected by flagging) and 50 cattle from the same pastureland in Nakaosobetsu, Hokkaido, Japan. Results revealed prevalence rates of 2.4% (2 of 85) in ticks and 2% (1 of 50) in cattle. The present study also reported the first molecular detection of the Anaplasma sp. closely related to A. phagocytophilum in Japan in H. douglasii, and established a new reliable PCR method that detects this Anaplasma sp. closely related to A. phagocytophilum in Japan.

Identification of Spotted Fever Group Rickettsia Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of the Sca4 Gene

DNA samples from four rickettsial species in Japan--R. japonica Aoki, R. asiatica IO-1, R. helvetica IP-1, and Rickettsia tamurae AT-1--were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in which a portion of the sca4 gene was amplified and restriction digested with the MspI enzyme. Each spotted fever group rickettsial species exhibited unique restriction profiles after MspI digestion. To examine the possibility of applying PCR-RFLP analysis to field samples, 36 Ixodes persulcatus ticks were collected from the Tokachi region (Hokkaido, Japan) by the flagging method and subjected to PCR-RFLP analysis. Of the ticks collected, five had identical restriction profiles to that of R. helvetica. Amplification and sequencing of the gltA gene for two of the five positive samples revealed that the sequences were identical to that of the Rickettsia sp. Tokachi-B-IP17m, which exhibits the highest similarity to R. helvetica. These results demonstrate that PCR-RFLP analysis is useful for identifying Rickettsia species in Japan and is applicable to epidemiological studies involving tick samples.